Optimization of Cas9 RNA sequence to reduce its unexpected effects as a microRNA sponge

Highlights Cas9 RNA functions as a miRNA sponge. Let-7 is the dominant regulated miRNA by Cas9 RNA. RNA sequence optimization of Cas9 by synonymous mutation improves its safety. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-022-01604-x.


Fig. S6
Basic expression level of let-7 target genes might be related to whether Cas9 regulates these genes by miRNA sponge mechanism (A) Volcano chart showed the different basic expression genes between " Let-7i-related Cas9-sensitive cells" and " Let-7i-related Cas9-insensitive cells".
Let-7i-related Cas9-sensitive cells: the top 35 types of cells in which more let-7i target genes were increased than decreased after Cas9 introduction. Let-7i-related Cas9-insensitive cells: the top 35 cell types in which more let-7i target genes were decreased than increased after Cas9 introduction. The basic gene expression levels in each cell line were mined from CCLE. These data showed there were some genes that have different basal expression levels between the " Let-7i-related Cas9-sensitive cells" and " Let-7i-related Cas9-insensitive cells"

(B) GO analysis result of the different basic expression genes between "
Let-7i-related Cas9-sensitive cells" and " Let-7i-related Cas9-insensitive cells". This data showed genes that have different basal expression levels between the two groups are not very significantly enriched in a specific pathway.
(C) GSEA analysis of all the validated let-7 target genes based on the different gene basic expression data between " Let-7i-related Cas9-sensitive cells" and " Let-7i-related Cas9-insensitive cells". And let-7 target genes were found to be negatively enriched in " Let-7i-related Cas9-sensitive cells".
(D) Heatmap showed the differently expressed target genes of miR-1-3p between "Let-7i-related Cas9-sensitive cells" and "Let-7i-related Cas9-insensitive". It showed there was no significant difference of the basic expression of these mir-1-3p target genes between the two groups of cells.
These data suggested that the basic expression levels of let-7 target genes are also related to whether they are regulated by Cas9, and if the expression level of target genes is too high, it is unlikely to further significantly up regulate such target genes through miRNA sponge mechanism. Let-7i-related Cas9-sensitive cells" and " Let-7i-related Cas9-insensitive cells".
Let-7i-related Cas9-sensitive cells: the top 35 types of cells in which more let-7i target genes were increased than decreased after Cas9 introduction.
Let-7i-related Cas9-insensitive cells: the top 35 cell types in which more let-7i target genes were decreased than increased after Cas9 introduction. The mutation data in each cell line were mined from CCLE.
(C) GO analysis of the 28 high-frequency mutant genes in all the 70 cell lines. Those whose mutation frequency is higher than 10% are considered to be high-frequency mutations. This data showed high-frequency mutant genes in the two groups are not significantly enriched in a specific pathway.
(D) Heatmap showed the number distribution of the 28 high-frequency mutant genes in the " Let-7i-related Cas9-insensitive cells" and " Let-7i-related Cas9-sensitive cells". The names of the reported let-7 target genes were marked purple.
(E) Venn diagram showed that among the 28 high-frequency mutant genes in the " Let-7i-related Cas9-insensitive cells" and " Let-7i-related Cas9-sensitive cells", half of the genes (14) are let-7 target, which is higher than the number (6) of the target genes of mirna-1-3p (a non-Cas9-miRNA).
(F) GO analysis of the 14 let-7 target genes that are also high-frequency mutant genes in all the 70 cell lines.
This result is very interesting, because almost all let-7 target genes with high-frequency mutations are distributed in the " Let-7i-related Cas9-insensitive cells".
These results suggested that, if some key let-7 target genes are mutated, the effect of Cas9 on up regulating let-7 target genes will be weakened.
On the other hand, in the " Let-7i-related Cas9-sensitive cells", there was almost no mutation distribution of these high-frequency mutant let-7 target genes, suggesting that if Cas9 is to play the role of up regulating let-7 target genes, some key let-7 target genes could not be mutated. (E) Let-7 target genes were selected from published datasets. The mRNA levels of these genes were detected by qPCR in Cas9-or control vector-transduced DU145 cells. The data are presented as the mean ± SD. **, p<0.01; t test, n=3.  (C) Immunohistochemistry analysis of KRAS, CDK6 and FN1 protein levels in tissues from the brain, pancreas and intestine of Cas9-transgenic mice and control mice. We also analyzed these genes in kidney, liver, lung, stomach, spine and bladder tissues of Cas9-transgenic and control mice and they were not significantly changed (data not shown). Cas9-Tg: Cas9-transgenic mouse.
In these in vivo experiments, we did not find strong evidence that Cas9 has a significant effect on mice. This may be due to the complexity of the organism and compensatory regulation, but it is worthy of more systematic and in-depth research in the future. For example, it is valuable to investigate whether there is a higher incidence of spontaneous cancer or any other disorder in their lifespan. Or a certain sample size of Cas9 transgenic mice may be exposed to tumorigenic factors, such as irradiation, diethylnitrosamine (DEN) or carbon tetrachloride (CCl4), and test whether Cas9-Tg mice have a higher tumor incidence than normal mice. Such experiments are worth pursuing in follow-up studies.   presented as the mean ± SD. *p < 0.05, t test; n = 3. according to the manufacturer's instructions.

Cell Counting Kit-8 (CCK-8) Assay
Transfected cells were counted and seeded into 96-well plates at a density of 10,000 cells/well. Detection of cell proliferation was performed after 24h. Cell proliferation was detected by a CCK-8 assay (Beyotime, China) according to the instructions of manufacturer. Α 10 μL volume of CCK-8 assay solution and an 100uL volume of medium were added to wells prior to incubation at 37°C for 1h in a cell incubator.
After the incubation, the 450nm absorbance of each well was determined by an Infinite 200 reader (TECAN). Each sample was performed in triplicate.

Cell Cycle Analysis
For cell cycle analysis, Cell Cycle and Apoptosis Analysis Kit (Beyotime, C1052, China) was used. Approximately 5x10 5 cells were collected and fixed in cold 75% alcohol at least 4 hours, and then incubated with 500ul of PI solution at 37℃ under dark condition for 30 min. About 1x10 4 cells were counted each time.

EdU Assay
Cells were trypsinized and added to the 96-well plates at a density of 2000 cells per well. After 24h, EdU kit (RIBOBIO, China) was used to measure cell proloferation according to the manufacturer's instructions.

Clonogenic Assays.
Cells were trypsinized and resuspended by 1mL of medium and then equal number of cells in each group were seeded in a well of 6-wells plate. To form the colonies, the cells were cultured in incubator for 8-10 days. Secondly, the colonies were washed by PBS three times, and fixed with 4% PFA about 20 min. After fixation, they were stained with 0.1% crystal violet (Sigma) about 30 min. Finally, the colonies were washed by PBS and counted.

Cell Transfection and Infection
Cultured cells were transfected with plasmid by Lipofectamine 3000 ( and 18S RNA or GAPDH mRNA was used as the endogenous control.

Western Blot
The total proteins of the cells were extracted by Whole Cell Lysis Assay (KeyGEN BioTECH, China) according to instructions of manufacturer. SDS-PAGE was done in 8-10%Tris-Glycine Gels (Invitrogen) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% BSA, incubated with primary and secondary antibodies, and washed according to standard procedures.

Bioinformatics analysis
The binding possibilities between Cas9 RNA and all the known human miRNAs were analyzed using miRanda software. The database with gene expression levels of 331 Cas9-introduced cell lines and their parental control counterparts can be downloaded from the following website：https://clue.io/data/XPR_BASE#CAS9_BASELINE. The differentially expressed genes in each cell were determined by t test.
The Cas9-miRNAs were those that could be predicted to bind Cas9 RNA (pLX311-Cas9 [1]， Addgene #118018， which has been used in the 331 cells dataset) under strict standards: the value of the score parameter was higher than 160, and the value of the energy parameter was lower than -25 kCal/Mol. The non-Cas9-miRNAs were those that could not be predicted to bind Cas9 RNA under loose parameters standards: threshold = 60, energy = -5kcal / mol, gap open penalty = -8, gap extend penalty = -2; from all the 2656 miRNAs downloaded from miRBase database, 2587 miRNAs contained gene binding sites in these loose parameters, and "No hit"" was found on the other 69 miRNAs. These 69 miRNAs were the non-Cas9-miRNAs.
The target genes of each miRNA were determined by the union of the prediction results of TargetScan and miRanda using the default parameter ： gap open penalty: -9.000000，Gap Extend Penalty: -4.000000，Score Threshold: 140.000000，Energy Threshold: -14.000000 kcal/mol，Scaling parameter: 4.000000.
The basic mRNA and miRNA expression data and the gene mutation data of 239 cell lines were mined in Cancer Cell Line Encyclopedia (CCLE, https://sites.broadinstitute.org/ccle ) .The GEO dataset GSE84534 was used to compare the mRNA expression levels between groups transduced with Cas9 (with gRNAs) or the control and was analyzed for all the let-7 target genes using GSEA.
The target genes of the let-7 miRNA family were found in miRTarBase with validation by at least two experimental methods and were used to construct the let-7 downstream geneset for GSEA of the GSE84534 dataset.

Animal experiments
The effect of Cas9 on the growth of tumors was determined using a xenograft model in male nude mice following reported protocols 12 .Each nude mouse received 1×10 7 cells in 100 μL serum-free medium blended with an equal volume of BD Matrigel Matrix (BD Biosciences). Cas9-transgenic mice were obtained from Shanghai Research Center for Southern Model Organisms. The serum general biochemical indicators in Cas9-transgenic mice and normal control mice were tested with an automatic biochemical analyzer. All animal experiments were performed following National Institutes of Health guidelines for animal treatment with approval from the animal subjects committee at Naval Medical University.

RNA-pulldown analysis
The RNA-pulldown analysis was performed using an immunoprecipitation kit (RNA-binding protein immunoprecipitation-assay kit for microRNA; MBL, Nagoya, Japan).In brief, HEK293 cells were transfected with biotin-labled-miR-let7-mimic or biotin-labled-miR-NC-mimic，and infected Cas9-expression or Cas9-mut-expression lentivirus for 48 hours. Different groups of cell lysate with RNA were pulled down using Streptavidin Magnetic Beads (MCE, USA). The streptavidin-bound RNA was extracted following the manufacturer's instructions and then subjected to qPCR analysis.

Statistical analysis
The data are presented as the means ± SD. Statistical comparisons between the experimental groups were analyzed using ANOVA or two-tailed Student's t test for the data obtained from the qPCR analyses, clonogenic analyses ，EdU analyses and gene expression assessment in each of the 331 cell types, and serum biomarkers assessment with an automatic biochemical analyzer and the luciferase activity assays.
P<0.05 or P<0.01 was considered to indicate statistical significance.